Foldover sensors and methods for making and using them

ABSTRACT

The invention disclosed herein includes sensors having three dimensional configurations that allow expansive “360°” sensing (i.e. sensing analyte from multiple directions) in the environments in which such sensors are disposed. Embodiments of the invention provide analyte sensors having foldable substrates adapted to produce optimized configurations of electrode elements as well as methods for making and using such sensors. Typical embodiments of the invention include glucose sensors used in the management of diabetes.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation application that claims the benefitunder 35 U.S.C. 120 of U.S. patent application Ser. No. 15/272,225,filed Sep. 21, 2016, which is a continuation application that claims thebenefit under 35 U.S.C. 120 of U.S. patent application Ser. No.13/779,271, filed Feb. 27, 2013, which claims priority under Section119(e) from U.S. Provisional Application Ser. No. 61/651,889, filed May25, 2012, the contents of which are incorporated herein by reference.

TECHNICAL FIELD

The invention relates to analyte sensors such as glucose sensors usefulin the management of diabetes.

BACKGROUND OF THE INVENTION

Electrochemical sensors are commonly used to detect or measure theconcentrations of in vivo analytes, such as glucose. Typically in suchanalyte sensing systems, an analyte (or a species derived from it) iselectro-active and generates a detectable signal at an electrode in thesensor. This signal is then correlated with the presence orconcentration of the analyte within a biological sample. In someconventional sensors, an enzyme is provided that reacts with the analyteto be measured, the byproduct of the reaction being qualified orquantified at the electrode. In one conventional glucose sensor,immobilized glucose oxidase catalyzes the oxidation of glucose to formhydrogen peroxide, which is then quantified by amperometric measurements(e.g. change in electrical current) through one or more electrodes.

In order to reduce the size of the sensors and/or increase theirsensitivity and efficiency, electrochemical sensors can be patternedwith multiple electrodes on both sides of the sensor. A variety ofelectrochemical sensors have also been developed to be multi-layered(e.g. double-sided), comprising multiple layers of electrodes andconductors interposed between multiple layers of dielectric materials.The electrochemical properties of multilayered sensors can be tailoredby altering certain design parameters (e.g. number of internal layers,layer thickness, area under the electrodes). However, fabricating suchsensors requires extra steps such as the patterning both/multiple sidesof sensor elements. Consequently, fabricating such multilayer sensorsrequires complicated and costly processes including, for example,reiteratively layering multiple elements. In addition, multilayersensors typically require the use of vias (vertical interception access)to establish vertical electrical connections between the differentlayers of conductors, elements which add to the cost and complexity offabricating such sensors.

There is a need for cost-effective sensors that provide the size,sensitivity, and efficiency advantages of double-sided and multilayersensors, as well as simplified manufacturing processes for fabricatingsuch sensors.

SUMMARY OF THE INVENTION

The invention disclosed herein includes sensors having three dimensionalconfigurations that allow expansive “360°” sensing (i.e. sensing analytefrom multiple directions) in the environments in which such sensors aredisposed. As discussed in detail below, sensors that provide suchexpansive sensing have advantages over sensors that obtain informationfrom a single location within a sensing environment. Embodiments of theinvention include amperometric analyte sensors formed from a foldablebase substrate as well as amperometric analyte sensors formed frommultiple base substrates that are adhered together. Such sensor designsprovide a number of advantageous characteristics in certain contexts,for example by facilitating sensor production processes as well asanalyte detection and/or characterization.

The invention disclosed herein has a number of embodiments. Anillustrative embodiment of the invention is an analyte sensor apparatuscomprising a base substrate comprising planar sheet of a flexiblematerial adapted to transition from a first configuration to a secondconfiguration when the base substrate is folded to form a fixed bend. Insuch embodiments of the invention, a working electrode, a counterelectrode and a reference electrode are disposed upon a first surface ofthe base substrate which is then folded to introduce fixed bends thatproduce specific sensor electrode configurations, for example, anelectrode configuration where at least one electrode is disposed on afirst side of the fixed bend; and at least one electrode is disposed ona second side of the fixed bend. Typically, other electronic elementsare disposed on the first surface of the base substrate, such as aplurality of contact pads and/or as a plurality of electrical conduitsadapted to transmit electrical signals between electrodes and contactpads.

As discussed in detail below, the base substrate can be made from avariety of materials and formed into a wide variety of shapes. Inillustrative working embodiments of the invention that are disclosedherein, the base substrate material can include a polymeric compositionsuch as a polyimide. In one working embodiment of the invention that isshown in FIG. 1, the base substrate is formed into a shape thatcomprises a rectangular body, a first longitudinal arm extending outwardfrom the rectangular body, and a second longitudinal arm extendingoutward from the rectangular body. In this illustrative workingembodiment, the first longitudinal arm and the second longitudinal armare parallel to each other. In certain embodiments of the invention,additional elements are used to facilitate base substrate manipulationand/or to stabilize a manipulated base architecture. Optionally forexample, a base substrate comprises a mark or other feature located inan area at which the base is folded in order to facilitate folding, forexample a demarcation, a perforation, or a kiss cut. In some embodimentsof the invention, the sensor apparatus comprises a locking member thatis adapted to inhibit movement of one or more elements that form or arecoupled to the folded base substrate (e.g. to inhibit movement of thefirst longitudinal arm or the second longitudinal arm). In someembodiments of the invention, the sensor apparatus comprises a spacingmember that is adapted to maintain a minimal distance between one ormore elements of the folded base substrate (e.g. the first longitudinalarm and the second longitudinal arm).

In typical embodiments of the invention, the sensor apparatus comprisesa plurality of working electrodes, for example, a first workingelectrode disposed on the first longitudinal arm and a second workingelectrode disposed on the second longitudinal arm (and/or multipleworking electrodes disposed on one or both longitudinal arm(s)). In someembodiments of the invention, the base substrate comprises a pluralityof reference electrodes, a plurality of working electrodes and aplurality of counter electrodes clustered together in units consistingessentially of one working electrode, one counter electrode and onereference electrode. Typically such clustered units are longitudinallydistributed on the base substrate in a repeating pattern of units. Intypical embodiments of the invention, the fixed bend in the basesubstrate configures the substrate in an architecture that results in atleast one electrode located on the first side of the fixed bend and atleast one electrode located on the second side of the fixed bend facingopposite directions.

Embodiments of the invention can include other structural elementsdesigned for use in specific analyte environments. For example, in someembodiments, the sensor is disposed within a housing (e.g. a tube) andadapted to be implanted in vivo (e.g. the tubed assembly embodimentshown in FIG. 6A). Typically, in such embodiments, the housing comprisesan aperture adapted to allow an aqueous medium in which the apparatus isdisposed to contact a working electrode. In alternative embodiments ofthe invention the apparatus does not comprise a housing that surroundsthe sensor (e.g. the tubeless assembly shown in FIG. 6B). In theseembodiments, the sensor is disposed within a needle adapted to pierce atissue and implant the apparatus in vivo. Typically, in suchembodiments, the needle is adapted to be removed from the tissuefollowing implantation of the analyte sensor apparatus.

Embodiments of the invention include further elements designed for usewith the folded sensors that are disclosed herein, for example thosethat are designed to analyze electrical signal data obtained fromelectrodes disposed on the folded base substrate. In some embodiments ofthe invention, the analyte sensor apparatus includes a processor and acomputer-readable program code having instructions, which when executed,cause the processor to assess electrochemical signal data obtained fromat least one working electrode and then compute analyte concentrationsbased upon the electrochemical signal data obtained from the workingelectrode. In certain embodiments of the invention, the processorcompares electrochemical signal data obtained from multiple workingelectrodes in order to, for example, adapt different electrodes to sensedifferent analytes, and/or to focus on different concentration ranges ofa single analyte; and/or to identify or characterize spurious sensorsignals (e.g. sensor noise, signals caused by interfering compounds andthe like) so as to enhance the accuracy of the sensor readings.

A related embodiment of the invention is a method of making a foldedanalyte sensor apparatus that is disclosed herein. Typically, suchmethods include the initial steps of providing a base substrate formedfrom a planar sheet of a flexible material having a first surface and asecond surface and adapted to transition from a first configuration to asecond configuration when the base substrate is folded. In the workingembodiments of the invention that are disclosed herein, the basesubstrate is designed to include a rectangular body, a firstlongitudinal arm extending outward from the rectangular body; and asecond longitudinal arm extending outward from the rectangular body.Typical embodiments of the invention include forming a plurality ofcontact pads and a plurality of electrical conduits upon the firstsurface of the base substrate. In such embodiments of the invention, theplurality of electrical conduits are of a size and formed from materialthat allows them to transmit electrical signals between electrodes andcontact pads separated by the fixed bend. These methods also include thesteps of forming a working electrode, a counter electrode and areference electrode on the first surface of the base substrate.Typically, at least one of these electrodes is formed on the firstlongitudinal arm and at least one other electrode is formed on thesecond longitudinal arm of the base substrate. These methods furtherinclude adding layers of materials onto one or more electrodes, forexample, forming an analyte sensing layer on the working electrode thatdetectably alters the electrical current at the working electrode in thepresence of an analyte as well as forming an analyte modulating layer onthe analyte sensing layer that modulates the diffusion of analytetherethrough. In certain embodiments of the invention, the analytesensing layer comprises glucose oxidase. Optionally, the analytemodulating layer comprises a hydrophilic polymer, for example a linearpolyurethane/polyurea polymer and/or a branched acrylate polymer.

Methods for making sensor embodiments of the invention include foldingthe base substrate so as to introduce a fixed bend that results in aconfiguration where at least one electrode is disposed on a first sideof the fixed bend, and at least one electrode is disposed on a secondside of the fixed bend. In this way, a folded analyte sensor embodimentof the invention can be formed. These methods can be used to produce awide variety of the folded sensor structures. For example, in someembodiments of the invention, the base substrate is formed so that thefirst longitudinal arm and the second longitudinal arm are parallel toeach other. Optionally in such embodiments, the base substrate is foldedso that the first longitudinal arm and the second longitudinal arm aresuperimposed on each other. In certain embodiments of the invention, thebase substrate is folded to introduce a fixed bend that configures thesubstrate in an orientation so that at least one electrode on the firstside of the fixed bend and at least one electrode on the second side ofthe fixed bend face opposite directions. In other embodiments of theinvention, the base substrate is folded so that the first side of thebase substrate that results from the fixed bend is in a plane is atleast 45 or 90 degrees off of the second side of the substrate thatresults from the fixed bend.

Embodiments of the invention are adapted for use with a variety ofelectrode configurations. For example, in some embodiments of theinvention, the sensor includes a single working electrode, counterelectrode and reference electrode formed on the base substrate. In otherembodiments of the invention, a plurality of working electrodes, counterelectrodes and reference electrodes clustered together in unitsconsisting essentially of one working electrode, one counter electrodeand one reference electrode are formed on the base substrate, and theclustered units are longitudinally distributed on at least onelongitudinal arm of the base substrate in a repeating pattern of units.In certain embodiments of the invention, one or more electrodes isformed an array of electrically conductive members disposed on the basesubstrate, the electrically conductive members are circular and have adiameter between 10 μm and 400 μm; and the array comprises at least 10electrically conductive members.

Yet another embodiment of the invention is a method of sensing ananalyte within the body of a mammal. Typically, this method comprisesimplanting an analyte sensor having a folded architecture within themammal (e.g. in the interstitial space of a diabetic individual),sensing an alteration in current at the working electrode in thepresence of the analyte; and then correlating the alteration in currentwith the presence of the analyte, so that the analyte is sensed. Whiletypical embodiments of the invention pertain to glucose sensors, thefolded sensor designs disclosed herein can be adapted for use with awide variety of devices known in the art.

Other objects, features and advantages of the present invention willbecome apparent to those skilled in the art from the following detaileddescription. It is to be understood, however, that the detaileddescription and specific examples, while indicating some embodiments ofthe present invention are given by way of illustration and notlimitation. Many changes and modifications within the scope of thepresent invention may be made without departing from the spirit thereof,and the invention includes all such modifications.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a top-down view illustrating one embodiment of a front surfaceof a foldover sensor having a first longitudinal member and a secondlongitudinal member each of which includes a three grouped units ofworking, counter and reference electrodes that are operably connected toa distal connection elements(s) by traces (for a comparison of thisembodiment to conventional, non-folded structures, see, e.g. FIG. 26 inU.S. Pat. No. 6,484,045). As shown in this figure, the base matrix has ashape that includes a rectangular body (500); a first longitudinal armextending outward from the rectangular body (520); and a secondlongitudinal arm (530) extending outward from the rectangular body. Inthis embodiment, the rectangular body and the longitudinal arms arelinked by a neck region (510).

FIGS. 2A-2C illustrate one embodiment of a foldover sensor. FIG. 2(a)shows the sensor prior to folding, FIG. 2(b) shows the front surface ofthe foldover sensor after folding, and FIG. 2(c) shows the back surfaceof the foldover sensor after folding.

FIGS. 3A-3D illustrate one embodiment of a sensor and graphed sensordata. FIG. 3(a) shows a top-down view of a foldover sensor prior tofolding over a longitudinal axis represented by a dotted line and FIG.3(b) shows a side view of the foldover sensor with the electrodes(black) facing out after folding. FIGS. 3C and 3D provide data fromstudies of such sensor structures in a In Vitro Testing System (BTS)that is designed to mimic in vivo conditions. In this system, sensorcurrent is measured periodically in the presence of known concentrationsof glucose and glucose values are then correlated with Isig, that issensor current (in nA). These graphs provide data (Isig over periods oftime) from experiments using sensors constructed to include in FIG.3(C): tubed sensors with the electrodes facing out and in FIG. 3(D)tubeless sensors with the electrodes facing out. In this system, sensorcurrent is measured periodically (typically once every five minutes) inthe presence of known concentrations of glucose and glucose values arethen correlated with ISIG, that is sensor current (in nA). When used inin vivo environments such as an interstitial space, these sensors can beused to measure glucose using calculations based on a formulaIG=ISIG×CAL, where IG is interstitial glucose value (in mmol/l ormg/dl), ISIG is sensor current (in nA) and CAL is calibration factor (inmmol/l/μA or mg/dl/μA).

FIGS. 4A-4B illustrate another embodiment of a sensor, with FIG. 4(a)showing a top-down view of a foldover sensor prior to folding over alongitudinal axis represented by a dotted line and FIG. 4(b) showing aside view of the foldover sensor with the electrodes (black) facing inafter folding.

FIGS. 5A-5C illustrate another embodiment of a sensor, with FIG. 5(a),showing a top-down view of a longitudinal member of a foldover sensorprior to folding over an axis of a longitudinal arm represented by adotted line perpendicular to its longitudinal direction; as well as FIG.5(b), showing a top-down view of the front surface of the foldoversensor after folding, and FIG. 5(c), showing a back surface foldoversensor after folding.

FIGS. 6A-6B illustrate embodiments of sensor elements, with FIG. 6(a)showing a tubed assembly comprising longitudinal members placed in tubeswith windows on both sides to increase circulation and allow fluidaccess to both sides of the longitudinal members and FIG. 6(b) showing,a tubeless assembly comprising longitudinal members held togetherthrough capillary action during assembly and free to separate afterimplementation.

FIGS. 7A-7B illustrate various embodiments of a sensor, with FIG. 7(a)showing a foldover sensor with electrodes only on the back surface andFIG. 7(b) showing a foldover sensor with a counter electrode on the backsurface and working electrodes on the front surface.

FIGS. 8A-8C illustrate embodiments of interlocking arms/members withFIG. 8(a) showing a hole in one longitudinal member; FIG. 8(b) showing acomplimentary flap in the other longitudinal member; and FIG. 8(c)showing an embodiment showing a spacer disposed on and/or between thefirst longitudinal arm and the second longitudinal arm.

FIG. 9 illustrates one embodiment of a foldover sensor having multiplesensing surfaces incorporated in a glucose sensor system.

FIG. 10 provides a perspective view illustrating a subcutaneous sensorinsertion set, a telemetered characteristic monitor transmitter device,and a data receiving device embodying features of the invention.

FIG. 11 shows a schematic of a potentiostat that may be used to measurecurrent in embodiments of the present invention. As shown in FIG. 11, apotentiostat 300 may include an op amp 310 that is connected in anelectrical circuit so as to have two inputs: Vset and Vmeasured. Asshown, Vmeasured is the measured value of the voltage between areference electrode and a working electrode. Vset, on the other hand, isthe optimally desired voltage across the working and referenceelectrodes. The current between the counter and reference electrode ismeasured, creating a current measurement (Isig) that is output from thepotentiostat.

FIGS. 12A-C show illustrations of amperometric analyte sensors formedfrom a plurality of planar layered elements. FIG. 12A shows anillustration of an electrode coated with various material layers. FIG.12B shows an illustration of a single sided sensor embodiment. FIG. 12Cshows an illustration of a double sided sensor embodiment. The substratedesign consists of the following layers in the embodiments shown inFIGS. 12B and 12C: a base polyimide (non-conducting) layer; ametallization layer patterned to form desired electronic elements; andan insulation polyimide (non-conducting) layer, patterned to formelectrode and contact pad designs.

FIG. 13A-13D show illustrations of a number of different electrode andelectronic element configurations useful with embodiments of theinvention. FIG. 13A1 shows a rectangular “traditional” electrodeconfiguration; FIG. 13A2 shows a microarray electrode configuration(with magnification of the microarray on left); FIG. 13B1 shows adistributed electrode configuration; FIG. 13B2 shows a micro-parallelelectrode configuration; FIG. 13C1 shows a 6-pin electrode design; FIG.13C2 shows a close up view of electrode arrangements in the lowerlongitudinal arm of the embodiment shown in FIG. 13C1; FIG. 13C3 shows aclose up view of electrode arrangements in the upper longitudinal arm ofthe embodiment shown in FIG. 13C1; and FIG. 13D1 shows a firstillustrative configuration of electrodes; FIG. 13D2 shows a secondillustrative configuration of electrodes; and FIG. 13D3 shows a thirdillustrative configuration of electrodes useful with embodiments of theinvention.

DETAILED DESCRIPTION OF THE INVENTION

Unless otherwise defined, all terms of art, notations, and otherscientific terms or terminology used herein are intended to have themeanings commonly understood by those of skill in the art to which thisinvention pertains. In some cases, terms with commonly understoodmeanings may be defined herein for clarity and/or for ready reference,and the inclusion of such definitions herein should not necessarily beconstrued to represent a substantial difference over what is generallyunderstood in the art. Many of the techniques and procedures describedor referenced herein are well understood and commonly employed usingconventional methodology by those skilled in the art.

All numbers recited in the specification and associated claims thatrefer to values that can be numerically characterized with a value otherthan a whole number (e.g. a distance) are understood to be modified bythe term “about”. Where a range of values is provided, it is understoodthat each intervening value, to the tenth of the unit of the lower limitunless the context clearly dictates otherwise, between the upper andlower limit of that range and any other stated or intervening value inthat stated range, is encompassed within the invention. The upper andlower limits of these smaller ranges may independently be included inthe smaller ranges, and are also encompassed within the invention,subject to any specifically excluded limit in the stated range. Wherethe stated range includes one or both of the limits, ranges excludingeither or both of those included limits are also included in theinvention. Furthermore, all publications mentioned herein areincorporated herein by reference to disclose and describe the methodsand/or materials in connection with which the publications are cited.Publications cited herein are cited for their disclosure prior to thefiling date of the present application. Nothing here is to be construedas an admission that the inventors are not entitled to antedate thepublications by virtue of an earlier priority date or prior date ofinvention. Further the actual publication dates may be different fromthose shown and require independent verification.

As discussed in detail below, embodiments of the invention relate to theuse of an electrochemical sensor that measures a concentration of ananalyte of interest or a substance indicative of the concentration orpresence of the analyte in fluid. In some embodiments, the sensor is acontinuous device, for example a subcutaneous, transdermal, orintravascular device. In some embodiments, the device can analyze aplurality of intermittent blood samples. The sensor embodimentsdisclosed herein can use any known method, including invasive, minimallyinvasive, and non-invasive sensing techniques, to provide an outputsignal indicative of the concentration of the analyte of interest.Typically, the sensor is of the type that senses a product or reactantof an enzymatic reaction between an analyte and an enzyme in thepresence of oxygen as a measure of the analyte in vivo or in vitro. Suchsensors typically comprise a membrane surrounding the enzyme throughwhich an analyte migrates. The product is then measured usingelectrochemical methods and thus the output of an electrode systemfunctions as a measure of the analyte.

Embodiments of the invention disclosed herein provide sensors of thetype used, for example, in subcutaneous or transcutaneous monitoring ofblood glucose levels in a diabetic patient. A variety of implantable,electrochemical biosensors have been developed for the treatment ofdiabetes and other life-threatening diseases. Many existing sensordesigns use some form of immobilized enzyme to achieve theirbio-specificity. Embodiments of the invention described herein can beadapted and implemented with a wide variety of known electrochemicalsensors elements, including for example, those disclosed in U.S. PatentApplication Nos. 20050115832, 20050008671, 20070227907, 20400025238,20110319734, 20110152654 and Ser. No. 13/707,400 filed Dec. 6, 2012,U.S. Pat. Nos. 6,001,067, 6,702,857, 6,212,416, 6,119,028, 6,400,974,6,595,919, 6,141,573, 6,122,536, 6,512,939 5,605,152, 4,431,004,4,703,756, 6,514,718, 5,985,129, 5,390,691, 5,391,250, 5,482,473,5,299,571, 5,568,806, 5,494,562, 6,120,676, 6,542,765, 7,033,336 as wellas PCT International Publication Numbers WO 01/58348, WO 04/021877, WO03/034902, WO 03/035117, WO 03/035891, WO 03/023388, WO 03/022128, WO03/022352, WO 03/023708, WO 03/036255, WO 03/036310 WO 08/042,625, andWO 03/074107, and European Patent Application EP 1153571, the contentsof each of which are incorporated herein by reference.

A. Illustrative Embodiments of the Invention and AssociatedCharacteristics Illustrative Embodiments

The invention disclosed herein includes sensors having three dimensionalconfigurations that allow expansive 360° sensing (i.e. sensing analytefrom multiple directions) in the environments in which such sensors aredisposed. As discussed in detail below, sensors that provide suchexpansive sensing have advantages over sensors that obtain informationfrom a single location within a sensing environment. Embodiments of theinvention include amperometric analyte sensors formed from a foldablebase substrate as well as amperometric analyte sensors formed frommultiple base substrates that are adhered together.

While the disclosure focuses primarily on embodiments of the inventionthat utilize foldable base substrates, those of skill in this technologyunderstand that this disclosure is readily adapted for use withembodiments of the invention that utilize two or more base substrates(e.g. as sensor element modules) that are adhered together. Such modulardouble-sided sensors can be made by overlaying or otherwise combiningtwo sensor substrates with active sensor electrodes to create a singleimplant sensor. Such double-sided sensors can be used to control theproximity of electrodes within an implant and/or their relativeproximity to each other. Moreover, such modular double-sided sensor canbe combined with the fold-over sensors disclosed herein to generatefurther sensor embodiments. Benefits of such modular sensors includegreater mechanical stability by doubling implant thickness whilesimultaneously avoiding the creation of a sensor that is too thick/stiff(and therefore prone to breaking).

Embodiments of the invention disclosed herein include amperometricanalyte sensors formed from a foldable base substrate. Such foldoversensor embodiments can be used as a means of putting the electrodes onthe opposite side of contact/bond pads without using vias, therebysimplifying the production process and reducing associated costs.Benefits of the foldover sensor include the selective positioning ofelectrical elements with minimal effort. Consequently, such embodimentsallow electrodes to be placed on both sides of a substrate, for exampleso that working and counter electrodes can be separated (e.g. so as tominimize interference from one electrode to another). Foldover sensorembodiments can also incorporate multiple working electrodes to getspatial separation between redundant electrodes that are designed tosense analytes such as glucose. In this way, such embodiments canovercome problems that can occur when a sensor electrode is disposedinto a localized suboptimal in vivo environment (e.g. localized scartissue and the like). Certain foldover sensor embodiments such as theone shown in FIG. 1 can be folded and placed into a needle that piercesa tissue in which the sensor is implanted. This allows a sensor to beinserted in vivo in a simple step. Moreover, in certain embodiment, twoarms of a base substrate can then flex outward from each other toincrease a separation distance in vivo. This flexing consequentlyincreases the spatial separation of sensor electrodes so as reduceproblematical issues such as those associated with localized analyteconcentration changes or tissue (e.g. scar tissue) effects.

As discussed below, sensor base substrates can be folded a number ofways to generate various embodiments of the invention. For example,sensor base substrates such as the embodiment shown in FIG. 1 can befolded back on itself at the tip to make assembly easier (see, e.g. FIG.5). Such embodiments can provide a desirable separation distance betweenelectrodes while allowing the tips to remain joined. In otherembodiments of the invention folding is used to reduce the length of asensor that is implanted in vivo. Moreover, in certain embodiments, onceinserted in vivo, the longitudinal arms of the sensor substrate sensorare free to flex and separate. In certain embodiments of the inventionwhere the arms flex outward and in to an in vitro environment, theflexed arms function as anchoring elements that inhibit sensor movementin the environment. This allows the sensor to be positioned deeper in atissue with a shorter implant. In addition, such pre-folding of thesensor may reduce the likelihood of sensor pullouts from the tissueenvironments in which they are disposed.

In typical sensor embodiments of the invention, a base substratecomprises a planar sheet of material having a first surface (e.g. thetop side of a sheet of material) and a second surface (e.g. a bottomside of the sheet of material). In these embodiments of the invention, aplurality of electrically conductive sensor elements includingelectrodes, electrical conduits and connecting regions are formed on asingle surface of the base structure, one which is typically made from amaterial such as a polyimide or other foldable polymeric substrate. Inillustrative embodiments of the invention disclosed herein, elements arefurther processed, for example, by cutting the base substrate, by theaddition of one or more layers of materials having selected functionalproperties (e.g. layers of a glucose oxidase composition) etc. Byforming and/or processing sensor elements on a single side of a foldablesheet of material, sensor production is simplified and made more costeffective. In addition, with such embodiments, sensor elements aredisposed in specific locations on the base structure so that thestructure can be precisely folded at specific locations in order tocreate specific three dimensional constellations of sensor elements,constellations designed to facilitate sensing in certain contexts, forexample, glucose sensing in in vivo tissues.

An illustrative embodiment of the invention is an analyte sensorapparatus comprising a base substrate formed from a planar sheet of aflexible material that is selected for its ability to transition from afirst configuration to a second configuration when the base substrate isfolded to form a fixed bend. In this embodiment of the invention, aworking electrode, a counter electrode and a reference electrode aredisposed upon a first surface of the base substrate. In suchembodiments, the base substrate is folded to introduce a fixed bend thatforms a specific 3-dimensional electrode configuration characterized inthat at least one electrode is disposed on a first side of the fixedbend; and at least one electrode is disposed on a second side of thefixed bend (e.g. a sensor base substrate can be folded so as to create afixed bend between counter and working electrodes).

As noted above, common embodiments of the invention comprise a specific3-dimensional electrode configuration characterized in that at least oneelectrode is disposed on a first side of the fixed bend and at least oneelectrode is disposed on a second side of the fixed bend. Embodimentsthat do not include at least one electrode on each side of a fixed bendare also contemplated, for example in a foldover configuration havingelectrodes disposed only on a single side of a fixed bend. In one suchembodiment of the invention, the base substrate can be folded, forexample, to change the direction of the electrodes (e.g. so as tooptimize the interaction with a sensing environment). In anotherembodiment of the invention having electrodes only on one side of afixed bend, the base substrate can be folded so as to impart mechanicalstability to the sensor when the sensor is implanted in vivo.

In typical embodiments of the invention, a plurality of contact pads arealso disposed upon the first surface of the base substrate along withthe electrodes, as well as a plurality of electrical conduits disposedupon the first surface of the base substrate. In such embodiments, theplurality of electrical conduits are adapted to transmit electricalsignals between electrodes and contact pads that are separated by thefixed bend. Typically in such embodiments, an analyte sensing layer isdisposed over the working electrode and includes one or more agents thatdetectably alter the electrical current at the working electrode in thepresence of an analyte (e.g. glucose oxidase); and an analyte modulatinglayer is then disposed over the analyte sensing layer that modulates thediffusion of analyte therethrough.

The base substrate of the sensor apparatus can be made from a variety ofmaterials and formed into a wide variety of shapes. In illustrativeworking embodiments of the invention such as the one shown in FIG. 1,the base substrate material can include a polymeric composition such asa polyimide and be formed (e.g. via laser cutting) into a shape thatcomprises a rectangular body, a first longitudinal arm extending outwardfrom the rectangular body, and a second longitudinal arm extendingoutward from the rectangular body. In this illustrative workingembodiment, the first longitudinal arm and the second longitudinal armare of the same length and parallel to each other. In other illustrativeembodiments, the first longitudinal arm and the second longitudinal armare disposed at an angle relative to each other and/or are of differentlengths.

Optionally, the base substrate further comprises an identifying markand/or a functional feature that facilitates folding, for example ademarcation, a perforation, or a kiss cut that helps a user identifyand/or manipulate the region at which the base substrate is folded. Insome embodiments of the invention, the sensor apparatus comprises alocking member that is adapted to inhibit movement of one or moreelements that form or are coupled to the folded base (e.g. to inhibitmovement of the first longitudinal arm or the second longitudinal arm).One illustrative embodiment of such a locking member is shown in FIGS.8A & 8B. In certain embodiments of the invention, the sensor apparatuscomprises a spacing member that is adapted to maintain a distancebetween the electrodes on the first longitudinal arm and the secondlongitudinal arm, so that the distance between the arms (or theelectrodes disposed on the arms) is at least 0.1, 0.2, 0.3, 0.4, 0.5,0.6, 0.7, 0.8, 0.9, 1, 1.25, 1.5, 1.75, 2, 2.5, 3, 3.5, 4, 4.5, 5, 6, 7,8, 9 or 10 millimeters. Optionally, the spacing member comprises acolumn of material (e.g. a rigid tubing of a defined length) that isdisposed on and/or spaced between the first longitudinal arm and thesecond longitudinal arm (see, e.g. FIG. 8C).

In typical embodiments of the invention, the apparatus comprises aplurality of working electrodes, for example, a first working electrodedisposed on the first longitudinal arm and a second working electrodedisposed on the second longitudinal arm (and/or multiple workingelectrodes is disposed on one longitudinal arm). In some embodiments ofthe invention, the base substrate comprises a plurality of referenceelectrodes, a plurality of working electrodes and a plurality of counterelectrodes clustered together in units consisting essentially of oneworking electrode, one counter electrode and one reference electrode.Optionally the clustered units are longitudinally distributed on thebase substrate in a repeating pattern of units. In such embodiments, oneworking electrode can be coated with a first set of layered materialsand another working electrode can be coated with a second set of layeredmaterials (e.g. different sets of materials that are designed to senseglucose in different concentration ranges). In certain embodiments ofthe invention, the fixed bend in the base substrate configures thesubstrate in an orientation so that at least one electrode on the firstside of the fixed bend and at least one electrode on the second side ofthe fixed bend face opposite directions (see, e.g. FIG. 3). Suchembodiments can be used for example to provide a greater distancebetween electrodes (e.g. counter and working electrodes), aconfiguration which may inhibit electron migration that can negativelyimpact sensor signals.

Embodiments of the invention can include other structural elementsdesigned for use in specific analyte environments. In some embodiments,at least a portion of the base substrate (e.g. the longitudinal arms ofa base substrate or the sensor electrodes that are located on such arms)are disposed within a housing (e.g. a tube) and adapted to be implantedin vivo (e.g. the “tubed” embodiment shown in FIG. 6A). Typically, insuch embodiments, the housing comprises an aperture adapted to allow anaqueous medium in which the apparatus is disposed to contact a workingelectrode. In such embodiments, sensors can be placed in tubes withapertures/windows on one or both sides to increase circulation and allowfluid access to both sides of the sensor. In alternative embodiments ofthe invention the apparatus does not comprise a housing that surrounds aportion of the base substrate (e.g. the “tubeless” embodiment shown inFIG. 6B). In such embodiments, sensor elements (e.g. longitudinal armsof a base substrate) can be held together through capillary actionduring assembly, while after implantation they are free to separate. Inthese embodiments, the sensor is disposed within a needle adapted topierce a tissue and implant the apparatus in vivo. Optionally in suchembodiments, the needle is adapted to be removed from the tissuefollowing implantation of the analyte sensor apparatus.

Embodiments of the invention include further elements designed for usewith the folded sensors that are disclosed herein, for example thosethat are designed to analyze electrical signal data obtained fromelectrodes disposed on the folded base substrate. In some embodiments ofthe invention, the analyte sensor apparatus includes a processor and acomputer-readable program code having instructions, which when executed,cause the processor to assess electrochemical signal data obtained fromat least one working electrode and then compute analyte concentrationsbased upon the electrochemical signal data obtained from the workingelectrode. In certain embodiments of the invention, the processorcompares electrochemical signal data obtained from multiple workingelectrodes in order to, for example, adapt different electrodes to sensedifferent analytes, and/or to focus on different concentration ranges ofa single analyte, and/or to identify or characterize spurious sensorsignals (e.g. sensor noise, signals caused by interfering compounds andthe like) so as to enhance the accuracy or reliability of the sensorreadings.

Related embodiments of the invention include methods of making a foldedanalyte sensor apparatus as disclosed herein. Briefly, in typicalmethods: (1) sensor electrodes and traces are patterned on to asubstrate formed from a polyimide or other flexible material; (2)chemistry layers are then applied to the electrodes (e.g. layerscomprising glucose oxidase, layers comprising a glucose limitingmembrane); and (3) the sensors are then laser cut and folded prior tofinal assembly, a step which results in electrodes disposed on the frontand back of the base substrate. Methods for making the sensors disclosedherein include the initial steps of providing a base substrate formedfrom a planar sheet of a flexible material having a first surface and asecond surface and adapted to transition from a first configuration to asecond configuration when the base substrate is folded. In the workingembodiments of the invention that are disclosed herein, the basesubstrate is designed to include a rectangular body, a firstlongitudinal arm extending outward from the rectangular body, and asecond longitudinal arm extending outward from the rectangular body. Inillustrative embodiments of the invention, the shape of the basesubstrate is formed by cutting the shape out of a sheet of material, forexample by laser cutting. In some embodiments of the invention, theelectrodes, contact pads, traces and the like are formed on thesubstrate before it is shaped into its final form. In other embodimentsof the invention, the electrodes, contact pads, traces and the like areformed on the substrate after it is shaped into its final form. FIG. 1provides an illustrative embodiment of a sensor substrate of theinvention. As shown in this figure, the base matrix has a shape thatincludes a rectangular body (500), a first longitudinal arm extendingoutward from the rectangular body (520); and a second longitudinal arm(530) extending outward from the rectangular body. In this embodiment,the rectangular body and the longitudinal arms are linked by a neckregion (510).

Typical embodiments of the invention include forming a plurality ofcontact pads and/or a plurality of electrical conduits upon the firstsurface of the base substrate. In such embodiments of the invention, theplurality of electrical conduits are selected to be of a size and formedfrom material that allows them to transmit electrical signals betweenelectrodes and contact pads separated by the architecture of the fixedbend (e.g. an amount of an electrically conductive material that willflex, not break when bent). In particular, in some embodiments of theinvention that were observed to exhibit unusual signal variation,deformations in the electrical conduits were observed in the regionswhere the conduits were folded. It is possible that these deformationsare associated with the observed electronic signal variation. The shape,size and material of these conduits is therefore tailored to thespecific architectures in which they are used (e.g. by increasing thewidth/girth/material of electrical conduits that are disposed overcomplex 3-dimensional architectures).

The methods of the invention include the steps of forming a workingelectrode, a counter electrode and a reference electrode on the firstsurface of the base substrate. Typically, at least one of theseelectrodes is formed on a first longitudinal arm and at least one otherelectrode is formed on a second longitudinal arm. These methods furtherinclude adding layers of materials onto one or more electrodes, forexample, forming an analyte sensing layer on the working electrode thatdetectably alters the electrical current at the working electrode in thepresence of an analyte as well as forming an analyte modulating layer onthe analyte sensing layer that modulates the diffusion of analytetherethrough. In certain embodiments of the invention, the analytesensing layer comprises glucose oxidase. In some embodiments of theinvention, the apparatus comprises an adhesion promoting layer disposedbetween the analyte sensing layer and the analyte modulating layer.Optionally, the analyte modulating layer comprises a hydrophiliccomb-copolymer having a central chain and a plurality of side chainscoupled to the central chain, wherein at least one side chain comprisesa silicone moiety.

Methods for making sensor embodiments of the invention can includefolding the base substrate so as to introduce a fixed bend that resultsin a configuration where at least one electrode is disposed on a firstside of the fixed bend, and at least one electrode is disposed on asecond side of the fixed bend. These methods can be used to produce awide variety of the folded sensor structures. For example, in someembodiments of the invention, the base substrate is formed so that thefirst longitudinal arm and the second longitudinal arm are parallel toeach other. Optionally, the base substrate is folded so that the firstlongitudinal arm and the second longitudinal arm are superimposed oneach other. In certain embodiments of the invention, the base substrateis folded to introduce a fixed bend that configures the substrate in anorientation so that at least one electrode on the first side of thefixed bend and at least one electrode on the second side of the fixedbend face opposite directions. In other embodiments of the invention,the base substrate is folded so that the first side of the basesubstrate that results from the fixed bend is in a plane is at least 40,50, 60, 70, 80 or 90 degrees off of the second side of the substratethat results from the fixed bend.

Embodiments of the invention are adapted for use with certain electrodeconfigurations. For example, in some embodiments of the invention, theworking electrode is formed as an array of electrically conductivemembers disposed on the base substrate, the electrically conductivemembers are circular and have a diameter between 10 μm and 400 μm; andthe array comprises at least 5, 10 or 15 electrically conductivemembers. In certain embodiments of the invention, a plurality of workingelectrodes, counter electrodes and reference electrodes clusteredtogether in units consisting essentially of one working electrode, onecounter electrode and one reference electrode are formed on the basesubstrate, and the clustered units are longitudinally distributed on atleast one longitudinal arm of the base substrate in a repeating patternof units. In some embodiments of the invention, a first clustered unitis disposed on a first longitudinal arm and a second clustered unit isdisposed on a second longitudinal arm.

As noted above, in embodiments of the invention, a base structure can beof a variety of shapes, depending upon the final constellation ofelements that is desired. Optionally, for example the base structure cancomprise a first longitudinal member and a second longitudinal member asshown for example in FIGS. 1-3. In embodiments such as those shown inFIGS. 1-3, the first and second longitudinal members each comprise atleast one electrode, and typically include a plurality of working,counter and reference electrodes (e.g. at least 2, 3, 4 or 5 groups)that are grouped together as a unit (e.g. at least 2, 3, 4 or 5 groupsof working, counter and reference electrodes) and positionallydistributed on a repeating pattern of units on the front surface (see,e.g. US. Patent Application Publication No. 2010/0025238, the contentsof which are incorporated herein by reference). As shown in FIG. 2, insome embodiments of the invention the electrodes are of different sizes,for example, a counter electrode that is at least 2× the size of aworking or reference electrode and/or a working electrode that is atleast 2× (or ½×) the size of a reference electrode. In anotherembodiment of the invention, the counter electrode is 2× the size of theworking electrode and the reference electrode is ⅓ the size of theworking electrode. In the embodiment shown in FIGS. 2A-2C the basestructure is folded along a longitudinal axis (dotted line in FIG. 2A)such that the first longitudinal member is substantially superimposedover the second longitudinal member. Typically, the base structurecomprises a dielectric composition. In common embodiments of theinvention, the folded sensor base substrates do not include vias,electrical connections in which must extend through dielectric layers toconnect conductive layers on either side of a dielectric material,thereby facilitating sensor production. In some embodiments of theinvention, first and second longitudinal members comprise substantiallysimilar electrical elements (see, e.g. FIG. 1 which shows both memberscomprising a plurality of working, counter and reference electrodespositionally distributed on a repeating pattern of units). In otherembodiments of the invention, first and second longitudinal memberscomprise substantially different electrical elements (see, e.g. FIG. 7which shows working electrode(s) on a first member and counterelectrodes on a second member).

As shown in FIGS. 2B-2C, in certain embodiments of the invention, thebase structure can be folded along a longitudinal axis such thatelectrodes on the first longitudinal arm/member are disposed in asubstantially opposite orientation or direction from electrodes on thesecond longitudinal member, for example so that the electroactivesurfaces of the electrodes are substantially oriented 180 degrees fromeach other (e.g. face away from each other as shown in FIG. 3B).Alternatively, the base structure can be folded along a longitudinalaxis such that the at least one electrode on the front surface of thefirst longitudinal member is disposed in the direction of at least oneelectrode on the front surface of the second longitudinal member (e.g.so that the electroactive surfaces of the electrodes substantially faceeach other as shown in FIG. 4B). Alternatively, the base structure canbe folded along a longitudinal axis such that the at least one electrodeon the front surface of the first longitudinal member is disposed in arelatively perpendicular orientation to at least one electrode on thefront surface of the second longitudinal member.

Embodiments of the invention can include a variety of differentconfigurations comprising bases of different shapes and sizes having oneor a plurality of folds (e.g. 2, 3, 4, 5, or more folds). As shown inFIG. 5, in certain embodiments of the invention, one or morelongitudinal members can be folded back on themselves, for example sothat electrodes are disposed on opposite sides of a single longitudinalmember. An illustrative embodiment of the invention comprises a foldoversensor including a base structure having a front surface and a backsurface, the base structure comprising at least one longitudinal member(and typically two or more), wherein the front surface of thelongitudinal member comprises at least one electrode (but typicallycomprises a plurality of working and/or reference, and/or counterelectrodes) and further is folded perpendicular to its longitudinaldirection or in a manner that orients a portion of the firstlongitudinal member over another portion of the first longitudinalmember (e.g. so that electrodes are disposed on opposite sides of alongitudinal member, see, e.g. FIG. 5).

In some embodiments of the invention, the composition of the basestructure is selected to have material properties that influence sensorconfiguration. Optionally in these embodiments, the base is formed from,or coated with, a dielectric material. For example, in certainembodiments of the invention, the base is made from a dielectricpolymeric material that is designed to flex in a certain directionfollowing the sensor fold and/or when the sensor is disposed in theenvironments in which an analyte is sensed. In one illustrative exampleshown in FIG. 6B, the material of the base structure can flex so as toincrease the distance between first and second longitudinal members onwhich the electrodes are disposed (e.g. so that the distance between apair of electrodes and/or the first and second longitudinal members isat 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 23, 4, 5, 6, 7 or 10millimeters). In addition, some embodiments of the invention includepositioning or locking members that facilitate proper sensorarchitecture. Optionally, for example, a first longitudinal member of abase structure comprises a first interlocking member and a secondlongitudinal member comprises a second interlocking member thatpositions the members in a specific orientation. In illustrativeembodiments, coupling the first interlocking member to the secondinterlocking member keeps the first longitudinal member substantiallysuperimposed over the second longitudinal member. FIG. 6 shows a sensorassembly where a tube is used to control sensor architecture. FIGS. 8A&Bshow an embodiment of an illustrative mechanism designed to position orlock portions of the sensor together (in this case the base material) soas to help maintain a desired sensor architecture.

As noted above, in embodiments of the invention, electrically conductivesensor elements such as electrodes and/or electrical conduits (e.g.traces) and/or connecting regions (e.g. contact pads) are formed on asingle surface of the base structure. In such embodiments, electricallyconducting sensor elements are disposed in specific locations on thebase structure so that the base can be subsequently folded at specificlocations in order to create a three dimensional constellation of sensorelements. In typical embodiments of the invention, the base structure isgenerally implemented as an electrically insulating (i.e.,non-conducting) material such as polyimide, rubber, TEFLON, MYLAR, andthe like. The base structure may be implemented using a wide varietyappropriate (or suitable) flexible dielectric materials known in the artdepending upon, for example, the architecture of a particular foldedsensor design. In embodiments of the invention, the materials used tomake the electrically conductive sensor elements and/or the structuresof these elements can be selected due to an ability to be amenable tofolding. For example, in addition to selecting optimized locations forthe folded elements, the length, thickness and/or width of theseelements (e.g. traces in a conductive path) as well as the number andspacing of the elements can be adapted for optimized functioning invarious three dimensional sensor architectures such as those disclosedherein. Typically, the electrical elements such as trace conductors canbe made from (i.e., produced from, implemented using, etc.) at least oneflexible electrically conductive material (e.g., Cu, Si, Cu, Al, Cr, Ti,Pt, Ir and the like). For example, trace conductors may be implementedusing any appropriate (or suitable) electrically conductive materialknown in the art depending upon, for example, the architecture of aparticular folded sensor design.

Embodiments of the invention include methods for making the foldoversensors disclosed herein. Such sensors can be made by adapting certainmethods known in the art, for example, those disclosed in U.S. Pat. No.6,484,045, the contents of which are incorporated by reference. Oneillustrative embodiment is method of making a foldover sensor, themethod comprising the steps of providing a base structure having a frontsurface and a back surface and then patterning a plurality ofelectrically conductive elements including at least one electrode (andoptionally a plurality of working counter and reference electrodesand/or a plurality of electrical conduits (e.g. traces and the like)and/or a plurality of contact pads and the like) on the front surface ofthe base structure. In such methods one can form the base structure intoa particular shape/geometry, for example by forming the base material ina mold and/or by cutting the base structure, for example to form a firstlongitudinal member and a second longitudinal member, each comprisingelectrically conductive elements. In such methods one can pattern theconductive elements onto specific regions of the base structure thatwill result in a specific three dimensional architecture when folded.

These methods can comprise folding the base structure to generate aconstellation of electrical elements having a specific three dimensionalarchitecture, for example by folding a base with longitudinal membersalong a longitudinal axis such that the first longitudinal member issubstantially superimposed over the second longitudinal member. In oneillustrative embodiment, the base structure is folded along alongitudinal axis such that the front surface of a first longitudinalmember faces in a substantially opposite direction away from the frontsurface of a second longitudinal member. Alternatively, the basestructure is folded along a longitudinal axis such that the frontsurface of the first longitudinal member faces towards the front surfaceof the second longitudinal member. Embodiments of the invention includeforming the sensor to include additional elements, for example anembodiment where a first longitudinal member comprises a firstinterlocking member and the second longitudinal member comprises secondinterlocking member complementary to the first interlocking member, andfurther comprising coupling the first interlocking member to the secondinterlocking member such that the first longitudinal member maintains aposition substantially superimposed over the second longitudinal member.Other embodiments of the invention include disposing the folded basestructure in a hollow tube (e.g. a needle, a catheter or the like).

Embodiments of the invention include methods of adding a plurality ofmaterials to the surface(s) of the electrode(s) disposed on the base,either prior to, or subsequent to folding (and sensors made from suchmethods). One such embodiment of the invention is a method of making asensor apparatus (e.g. a glucose sensor) for implantation within amammal comprising the steps of: providing a base substrate; forming aconductive layer on the base substrate, wherein the conductive layerincludes an electrode (and typically a working electrode, a referenceelectrode and a counter electrode); forming an analyte sensing layer onthe conductive layer, wherein the analyte sensing layer includes acomposition that can alter the electrical current at the electrode inthe conductive layer in the presence of an analyte (e.g. glucoseoxidase); optionally forming a protein layer over the analyte sensinglayer; forming an adhesion promoting layer on the analyte sensing layeror the optional protein layer; forming an analyte modulating layerdisposed on the adhesion promoting layer, wherein the analyte modulatinglayer includes a composition that modulates the diffusion of the analytetherethrough; and forming a cover layer disposed on at least a portionof the analyte modulating layer, wherein the cover layer furtherincludes an aperture over at least a portion of the analyte modulatinglayer. In different embodiments of the invention, the base material canbe folded following the application of a specific material, for examplean analyte modulating layer, a cover layer, etc. See, e.g. U.S. PatentPublication No. 2010/0025238, the contents of which are incorporated byreference.

In some embodiments of the invention, the base structure comprises afoldable yet rigid and flat structure suitable for use inphotolithographic mask and etch processes. In this regard, the basestructure typically includes at least one surface having a high degreeof uniform flatness. Base structure materials can include, for example,metals such as stainless steel, aluminum and nickel titanium memoryalloys (e.g. NITINOL) as well as polymeric/plastic materials such asdelrin, etc. Base structure materials can be made from, or coated with,a dielectric material. In some embodiments, the base structure isnon-rigid and can be a layer of film or insulation that is used as asubstrate for patterning electrical elements (e.g. electrodes, tracesand the like), for example plastics such as polyimides and the like. Aninitial step in the methods of the invention typically includes theformation of a base substrate of the sensor. Optionally the planar sheetof material is formed and/or disposed on a support such as a glass orceramic plate during sensor production (see, e.g. FIG. 2A). The basestructure can be disposed on a support (e.g. a glass plate) by anydesired means, for example by controlled spin coating. Optionally, abase substrate layer of insulative material is formed on the support,typically by applying the base substrate material onto the support inliquid form and thereafter spinning the support to yield a basesubstrate structure that is thin and of a substantially uniformthickness. These steps can be repeated to build up a base substratestructure to a desired thickness. This can then be followed by asequence of photolithographic and/or chemical mask and etch steps toform the electrically conductive components. In an illustrative form,the base substrate comprises a thin film sheet of insulative material,such as a polyimide substrate that is used to pattern electricalelements. The base substrate structure may comprise one or more of avariety of elements including, but not limited to, carbon, nitrogen,oxygen, silicon, sapphire, diamond, aluminum, copper, gallium, arsenic,lanthanum, neodymium, strontium, titanium, yttrium, or combinationsthereof.

The methods of the invention further include the generation of anelectrically conductive layer on the base substrate that function as oneor more sensing elements. Typically these sensing elements includeelectrodes, electrical conduits (e.g. traces and the like), contact padsand the like that are formed by one of the variety of methods known inthe art such as photolithography, etching and rinsing to define thegeometry of the active electrodes. The electrodes can then be madeelectrochemically active, for example by electrodeposition of Pt blackfor the working and counter electrode, and silver followed by silverchloride on the reference electrode. A sensor layer such as a analytesensing enzyme layer can then be disposed on the sensing layer byelectrochemical deposition or a method other than electrochemicaldeposition such a spin coating, followed by vapor crosslinking, forexample with a dialdehyde (glutaraldehyde) or a carbodi-imide.

In an exemplary embodiment of the invention, the base substrate isinitially coated with a thin film conductive layer by electrodedeposition, surface sputtering, or other suitable patterning or otherprocess step. In one embodiment this conductive layer may be provided asa plurality of thin film conductive layers, such as an initialchrome-based layer suitable for chemical adhesion to a polyimide basesubstrate followed by subsequent formation of thin film gold-based andchrome-based layers in sequence. In alternative embodiments, otherelectrode layer conformations or materials can be used. The conductivelayer is then covered, in accordance with conventional photolithographictechniques, with a selected photoresist coating, and a contact mask canbe applied over the photoresist coating for suitable photoimaging. Thecontact mask typically includes one or more conductor trace patterns forappropriate exposure of the photoresist coating, followed by an etchstep resulting in a plurality of conductive sensor traces remaining onthe base substrate. In an illustrative sensor construction designed foruse as a subcutaneous glucose sensor, each sensor trace can include twoor three parallel sensor elements corresponding with two or threeseparate electrodes such as a working electrode, a counter electrode anda reference electrode.

Additional functional coatings or cover layers can then be applied to anelectrode or other senor element by any one of a wide variety of methodsknown in the art, such as spraying, dipping, etc. Some embodiments ofthe present invention include an analyte modulating layer deposited overa enzyme-containing layer that is disposed over a working electrode. Inaddition to its use in modulating the amount of analyte(s) that contactsthe active sensor surface, by utilizing an analyte limiting membranelayer, the problem of sensor fouling by extraneous materials is alsoobviated. As is known in the art, the thickness of the analytemodulating membrane layer can influence the amount of analyte thatreaches the active enzyme. Consequently, its application is typicallycarried out under defined processing conditions, and its dimensionalthickness is closely controlled. Microfabrication of the underlyinglayers can be a factor which affects dimensional control over theanalyte modulating membrane layer as well as exact the composition ofthe analyte limiting membrane layer material itself. In this regard, ithas been discovered that several types of copolymers, for example, acopolymer of a siloxane and a nonsiloxane moiety, are particularlyuseful. These materials can be microdispensed or spin-coated to acontrolled thickness. Their final architecture may also be designed bypatterning and photolithographic techniques in conformity with the otherdiscrete structures described herein.

In some embodiments of the invention, the sensor is made by methodswhich apply an analyte modulating layer that comprises a hydrophilicmembrane coating which can regulate the amount of analyte that cancontact the enzyme of the sensor layer. For example, a cover layer thatis added to the glucose sensing elements of the invention can comprise aglucose limiting membrane, which regulates the amount of glucose thatcontacts glucose oxidase enzyme layer on an electrode. Such glucoselimiting membranes can be made from a wide variety of materials known tobe suitable for such purposes, e.g., silicones such as polydimethylsiloxane and the like, polyurethanes, cellulose acetates, Nafion,polyester sulfonic acids (e.g. Kodak AQ), hydrogels or any othermembrane known to those skilled in the art that is suitable for suchpurposes. In certain embodiments of the invention, the analytemodulating layer comprises a hydrophilic polymer. In some embodiments ofthe invention the analyte modulating layer comprises a linearpolyurethane/polyurea polymer and/or a branched acrylate polymer, and/ora mixture of such polymers.

In some embodiments of the methods of invention, an adhesion promoterlayer is disposed between a cover layer (e.g. an analyte modulatingmembrane layer) and a analyte sensing layer in order to facilitate theircontact and is selected for its ability to increase the stability of thesensor apparatus. As noted herein, compositions of the adhesion promoterlayer are selected to provide a number of desirable characteristics inaddition to an ability to provide sensor stability. For example, somecompositions for use in the adhesion promoter layer are selected to playa role in interference rejection as well as to control mass transfer ofthe desired analyte. The adhesion promoter layer can be made from anyone of a wide variety of materials known in the art to facilitate thebonding between such layers and can be applied by any one of a widevariety of methods known in the art.

The finished sensors produced by such processes are typically quicklyand easily removed from a support structure (if one is used), forexample, by cutting along a line surrounding each sensor on the supportstructure. The cutting step can use methods typically used in this artsuch as those that include a laser cutting device that is used to cutthrough the base and cover layers and the functional coating layersalong a line surrounding or circumscribing each sensor, typically in atleast slight outward spaced relation from the conductive elements sothat the sufficient interconnected base and cover layer material remainsto seal the side edges of the finished sensor. Since the base substrateis typically not physically attached or only minimally adhered directlyto the underlying support, the sensors can be lifted quickly and easilyfrom the support structure, without significant further processing stepsor potential damage due to stresses incurred by physically pulling orpeeling attached sensors from the support structure. The supportstructure can thereafter be cleaned and reused, or otherwise discarded.The functional coating layer(s) can be applied either before or afterother sensor components are removed from the support structure (e.g. bycutting).

Embodiments of the invention include methods of sensing an analyte (e.g.glucose) within the body of a mammal (e.g. a diabetic patient), themethod comprising implanting a foldover analyte sensor embodimentdisclosed herein into an in vivo environment and then sensing one ormore electrical fluctuations such as alteration in current at theworking electrode and correlating the alteration in current with thepresence of the analyte, so that the analyte is sensed. Typically, thismethod comprises implanting a glucose sensor having a foldedarchitecture within the interstitial space of a diabetic individual,sensing an alteration in current at the working electrode in thepresence of glucose; and then correlating the alteration in current withthe presence of the glucose, so that glucose is sensed. While typicalembodiments of the invention pertain to glucose sensors, the foldedsensor designs disclosed herein can be adapted for use with a widevariety of devices known in the art.

As discussed in detail below, embodiments of the invention includesensor systems comprising addition elements designed to facilitatesensing of an analyte. For example, in certain embodiments of theinvention, the base material comprising the sensor electrodes isdisposed within a housing (e.g. a lumen of a catheter) and/or associatedwith other components that facilitate analyte (e.g. glucose) sensing.FIG. 6A shows an embodiment of the invention comprising a hollow tube(e.g. a catheter) housing the base structure. FIG. 9 shows anotherembodiment of a foldover sensor combined with other components useful inin vivo glucose sensor system embodiments. One illustrative foldoversensor system comprises a processor, a base comprising a firstlongitudinal member and a second longitudinal member, the first andsecond longitudinal members each comprising at least one electrodehaving an electrochemically reactive surface, wherein theelectrochemically reactive surface generates an electrochemical signalthat is assessed by the processor in the presence of an analyte; and acomputer-readable program code having instructions, which when executedcause the processor to assess electrochemical signal data obtained fromthe electrodes; and compute an analyte presence or concentration basedupon the electrochemical signal data obtained from the electrode. Inthis system, the base of the sensor is folded longitudinally such thatthe first longitudinal member substantially overlaps the secondlongitudinal member. Embodiments of the invention described herein canalso be adapted and implemented with amperometric sensor structures, forexample those disclosed in U.S. Patent Application Publication Nos.20070227907, 20400025238, 20110319734 and 20110152654, the contents ofeach of which are incorporated herein by reference.

Illustrative Characteristics of Embodiments of the Invention

The 360° sensor designs that are disclosed herein are designed toaddress a number of problematical issues that can occur in certainconventional sensor designs. For example, certain continuous glucosemonitoring systems involve the use of a single sensor that must becalibrated against a reference value at regular intervals. In suchembodiments, system accuracy is dependent upon the output of thisindividual sensor and may be affected by transient periods of sensorinstability. The reliability of such sensor systems can be improved ifoutputs multiple sensing electrodes are utilized. However, conventionalsensor designs that incorporate multiple sensing electrodes typicallyrequire additional and costly manufacturing steps. As noted below,embodiments of the invention overcome these problems in this technology.

The simple redundancy provided by embodiments of the invention, namelythose that include multiple working electrodes (e.g. those havingidentical layers of material layers) can be used to address a number ofproblematical issues that can occur in certain conventional sensordesigns. For example, in certain embodiments of the invention, the dataobtained from multiple working electrodes can be combined in real-timeor during post-processing to enhance sensor reliability. In thiscontext, a number of methods can be used for combining raw outputs fromtwo or more redundant sensors. In one illustrative embodiment, rawvalues from redundant electrodes are averaged to generate a singleoutput before calculating the final sensor glucose value. In anotherillustrative embodiment, sensor algorithms can be employed which analyzeraw data from individual and multiple working electrodes in order toidentify fault conditions (e.g. Electrochemical Impedance Check, noise,drift etc.). In such embodiments, only raw data from the uncompromisedelectrodes is then used for the final analyte determinations.

Embodiments of the invention are also useful in glucose diagnosticsensing applications. For example, a multi-electrode glucose sensorsystem can also be used to improve the decisions made by the devicealgorithm (thus reliability) by providing additional information on thesensing environment. In such embodiments, different electrode layerchemistries are deposited and/or different electrode potentials areapplied to different electrodes, for example those that are differentfrom those used for glucose sensing, such as a working electrode run at−650 mV as opposed to 535 mV (which can be instead used only for glucosesensing) in order to characterize factors associated with glucose sensorreliability including background noise, the presence or concentrationsof interfering species, oxygen concentrations or the pH of anenvironment in which a glucose sensor is placed.

Embodiments of the invention are also useful to increase the reliabilityof glucose sensor measurements in diabetic patient hyperglycemic and/orhypoglycemic blood glucose concentration ranges. For example, in certainembodiments of the invention, individual electrodes can be used toobtain higher accuracy in specific hyperglycemic and/or hypoglycemicregions. In this context, a multi-working electrode sensor can alsoprovide the bandwidth for specific designs that can provide highlyreliable data at specific hyperglycemic and/or hypoglycemic ranges. Thiscan be accomplished, for example, by optimizing the electrode sizes ordesigns. In particular, a smaller working electrode generally showsreduced drift, better linearity and low background. However, the limitedsignal magnitude with such smaller electrodes can limit sensor accuracyat certain hyperglycemic ranges (high glucose levels). Similarly, alarger working electrode typically shows more noise and higherbackground at hypoglycemic ranges (low glucose levels). However, suchlarger electrodes can give a higher dynamic range for hyperglycemicsensitivity. In embodiments of the invention comprising a multi-workingelectrode system, these two or more electrodes can be combined into asingle sensor in order to obtain the optimal hypoglycemic andhyperglycemic range information from each working electrode of adifferent size.

Embodiments of the invention are also useful to optimize glucose sensorperformance based on factors specific to the amount of time after sensorimplantation that glucose is sensed. For example, embodiments of theinvention can be used to assess the performance and/or increase thereliability of sensors used in early wear (i.e. the first 24 hours orday 1) performance and late wear (e.g. days 7-10) performance by usingsensors having working electrodes upon which selective chemistrydesigned for either early wear performance or late wear performance isdisposed. For example, glucose sensors having working electrodes uponwhich thinner layers of materials are deposited (e.g. glucose oxidase, aglucose limiting membrane etc.) are observed to produce more accuratereadings in early wear, but tend to lose sensitivity after day 2. Athinner or high-permeable chemistry may hydrate quickly for improved day1 accuracy but may not be ideal for long term wear. In contrast, glucosesensors having working electrodes upon which thicker layers of materials(e.g. an analyte sensing layer, an analyte modulating layer etc.) aredeposited are observed to exhibit stability and reliability during laterwear but not at start-up (early wear). For example, a thicker orlow-permeable chemistry may hydrate more slowly compromising day 1accuracy but provide long-term sensitivity (improved later wearaccuracy). Consequently, by selectively controlling the properties ofthe materials disposed on a working electrode, (e.g. concentrations ofreagents, thickness, permeability) one can to optimize sensorperformance based on time after implantation. In this context, themulti-electrode systems disclosed herein allow dedicated electrodes tohave specialized material layers that are designed to optimize earlyand/or late wear sensor accuracy.

B. Illustrative Analyte Sensor Constituents Used in Embodiments of theInvention

The following disclosure provides examples of typicalelements/constituents used in sensor embodiments of the invention. Whilethese elements can be described as discreet units (e.g. layers), thoseof skill in the art understand that sensors can be designed to containelements having a combination of some or all of the material propertiesand/or functions of the elements/constituents discussed below (e.g. anelement that serves both as a supporting base constituent and/or aconductive constituent and/or a matrix for the analyte sensingconstituent and which further functions as an electrode in the sensor).Those in the art understand that these thin film analyte sensors can beadapted for use in a number of sensor systems such as those describedbelow.

Base Constituent

Sensors of the invention typically include a base constituent (see, e.g.element 402 in FIG. 12). The term “base constituent” is used hereinaccording to art accepted terminology and refers to the constituent inthe apparatus that typically provides a supporting matrix for theplurality of constituents that are stacked on top of one another andcomprise the functioning sensor. In one form, the base constituentcomprises a thin film sheet of insulative (e.g. electrically insulativeand/or water impermeable) material. This base constituent can be made ofor coated with a wide variety of materials having desirable qualitiessuch as dielectric properties, water impermeability and hermeticity.Some materials include metallic, and/or ceramic and/or polymericsubstrates or the like. Embodiments of the invention utilize basesubstrates formed from flexible material(s) selected for an ability totransition from a first configuration to a second configuration when thebase substrate is folded to form a fixed bend. Such materials must beflexible enough to bend but not break when folded. At the same time,such materials must be stiff/rigid enough to form a fixed (permanent)bend when folded.

Conductive Constituent

The electrochemical sensors of the invention typically include aconductive constituent disposed upon the base constituent that includesat least one electrode for contacting an analyte or its byproduct (e.g.oxygen and/or hydrogen peroxide) to be assayed (see, e.g. element 404 inFIG. 12). The term “conductive constituent” is used herein according toart accepted terminology and refers to electrically conductive sensorelements such as electrodes, contact pads, traces and the like. Anillustrative example of this is a conductive constituent that forms aworking electrode that can measure an increase or decrease in current inresponse to exposure to a stimuli such as the change in theconcentration of an analyte or its byproduct as compared to a referenceelectrode that does not experience the change in the concentration ofthe analyte, a coreactant (e.g. oxygen) used when the analyte interactswith a composition (e.g. the enzyme glucose oxidase) present in analytesensing constituent 410 or a reaction product of this interaction (e.g.hydrogen peroxide). Illustrative examples of such elements includeelectrodes which are capable of producing variable detectable signals inthe presence of variable concentrations of molecules such as hydrogenperoxide or oxygen.

In addition to the working electrode, the analyte sensors of theinvention typically include a reference electrode or a combinedreference and counter electrode (also termed a quasi-reference electrodeor a counter/reference electrode). If the sensor does not have acounter/reference electrode then it may include a separate counterelectrode, which may be made from the same or different materials as theworking electrode. Typical sensors of the present invention have one ormore working electrodes and one or more counter, reference, and/orcounter/reference electrodes. One embodiment of the sensor of thepresent invention has two, three or four or more working electrodes.These working electrodes in the sensor may be integrally connected orthey may be kept separate. Optionally, the electrodes can be disposed ona single surface or side of the sensor structure. Alternatively, theelectrodes can be disposed on a multiple surfaces or sides of the sensorstructure. In certain embodiments of the invention, the reactivesurfaces of the electrodes are of different relative areas/sizes, forexample a 1× reference electrode, a 2.6× working electrode and a 3.6×counter electrode.

Interference Rejection Constituent

The electrochemical sensors of the invention optionally include aninterference rejection constituent disposed between the surface of theelectrode and the environment to be assayed. In particular, certainsensor embodiments rely on the oxidation and/or reduction of hydrogenperoxide generated by enzymatic reactions on the surface of a workingelectrode at a constant potential applied. Because amperometricdetection based on direct oxidation of hydrogen peroxide requires arelatively high oxidation potential, sensors employing this detectionscheme may suffer interference from oxidizable species that are presentin biological fluids such as ascorbic acid, uric acid and acetaminophen.In this context, the term “interference rejection constituent” is usedherein according to art accepted terminology and refers to a coating ormembrane in the sensor that functions to inhibit spurious signalsgenerated by such oxidizable species which interfere with the detectionof the signal generated by the analyte to be sensed. Certaininterference rejection constituents function via size exclusion (e.g. byexcluding interfering species of a specific size). Examples ofinterference rejection constituents include one or more layers orcoatings of compounds such as hydrophilic polyurethanes, celluloseacetate (including cellulose acetate incorporating agents such aspoly(ethylene glycol), polyethersulfones, polytetra-fluoroethylenes, theperfluoronated ionomer Nafion™, polyphenylenediamine, epoxy and thelike.

Analyte Sensing Constituent

The electrochemical sensors of the invention include an analyte sensingconstituent disposed on the electrodes of the sensor (see, e.g. element410 in FIG. 12). The term “analyte sensing constituent” is used hereinaccording to art accepted terminology and refers to a constituentcomprising a material that is capable of recognizing or reacting with ananalyte whose presence is to be detected by the analyte sensorapparatus. Typically, this material in the analyte sensing constituentproduces a detectable signal after interacting with the analyte to besensed, typically via the electrodes of the conductive constituent. Inthis regard the analyte sensing constituent and the electrodes of theconductive constituent work in combination to produce the electricalsignal that is read by an apparatus associated with the analyte sensor.Typically, the analyte sensing constituent comprises an oxidoreductaseenzyme capable of reacting with and/or producing a molecule whose changein concentration can be measured by measuring the change in the currentat an electrode of the conductive constituent (e.g. oxygen and/orhydrogen peroxide), for example the enzyme glucose oxidase. An enzymecapable of producing a molecule such as hydrogen peroxide can bedisposed on the electrodes according to a number of processes known inthe art. The analyte sensing constituent can coat all or a portion ofthe various electrodes of the sensor. In this context, the analytesensing constituent may coat the electrodes to an equivalent degree.Alternatively, the analyte sensing constituent may coat differentelectrodes to different degrees, with for example the coated surface ofthe working electrode being larger than the coated surface of thecounter and/or reference electrode.

Typical sensor embodiments of this element of the invention utilize anenzyme (e.g. glucose oxidase) that has been combined with a secondprotein (e.g. albumin) in a fixed ratio (e.g. one that is typicallyoptimized for glucose oxidase stabilizing properties) and then appliedon the surface of an electrode to form a thin enzyme constituent. In atypical embodiment, the analyte sensing constituent comprises a GOx andHSA mixture. In a typical embodiment of an analyte sensing constituenthaving GOx, the GOx reacts with glucose present in the sensingenvironment (e.g. the body of a mammal) and generates hydrogen peroxide.

As noted above, the enzyme and the second protein (e.g. an albumin) aretypically treated to form a crosslinked matrix (e.g. by adding across-linking agent to the protein mixture). As is known in the art,crosslinking conditions may be manipulated to modulate factors such asthe retained biological activity of the enzyme, its mechanical and/oroperational stability. Illustrative crosslinking procedures aredescribed in U.S. patent application Ser. No. 10/335,506 and PCTpublication WO 03/035891 which are incorporated herein by reference. Forexample, an amine cross-linking reagent, such as, but not limited to,glutaraldehyde, can be added to the protein mixture. The addition of across-linking reagent to the protein mixture creates a protein paste.The concentration of the cross-linking reagent to be added may varyaccording to the concentration of the protein mixture. Whileglutaraldehyde is an illustrative crosslinking reagent, othercross-linking reagents may also be used or may be used in place ofglutaraldehyde. Other suitable cross-linkers also may be used, as willbe evident to those skilled in the art.

As noted above, in some embodiments of the invention, the analytesensing constituent includes an agent (e.g. glucose oxidase) capable ofproducing a signal (e.g. a change in oxygen and/or hydrogen peroxideconcentrations) that can be sensed by the electrically conductiveelements (e.g. electrodes which sense changes in oxygen and/or hydrogenperoxide concentrations). However, other useful analyte sensingconstituents can be formed from any composition that is capable ofproducing a detectable signal that can be sensed by the electricallyconductive elements after interacting with a target analyte whosepresence is to be detected. In some embodiments, the compositioncomprises an enzyme that modulates hydrogen peroxide concentrations uponreaction with an analyte to be sensed. Alternatively, the compositioncomprises an enzyme that modulates oxygen concentrations upon reactionwith an analyte to be sensed. In this context, a wide variety of enzymesthat either use or produce hydrogen peroxide and/or oxygen in a reactionwith a physiological analyte are known in the art and these enzymes canbe readily incorporated into the analyte sensing constituentcomposition. A variety of other enzymes known in the art can produceand/or utilize compounds whose modulation can be detected byelectrically conductive elements such as the electrodes that areincorporated into the sensor designs described herein. Such enzymesinclude for example, enzymes specifically described in Table 1, pages15-29 and/or Table 18, pages 111-112 of Protein Immobilization:Fundamentals and Applications (Bioprocess Technology, Vol 14) by RichardF. Taylor (Editor) Publisher: Marcel Dekker; Jan. 7, 1991) the entirecontents of which are incorporated herein by reference.

Protein Constituent

The electrochemical sensors of the invention optionally include aprotein constituent disposed between the analyte sensing constituent andthe analyte modulating constituent (see, e.g. element 416 in FIG. 12).The term “protein constituent” is used herein according to art acceptedterminology and refers to constituent containing a carrier protein orthe like that is selected for compatibility with the analyte sensingconstituent and/or the analyte modulating constituent. In typicalembodiments, the protein constituent comprises an albumin such as humanserum albumin. The HSA concentration may vary between about 0.5%-30%(w/v). Typically, the HSA concentration is about 1-10% w/v, and mosttypically is about 5% w/v. In alternative embodiments of the invention,collagen or BSA or other structural proteins used in these contexts canbe used instead of or in addition to HSA. This constituent is typicallycrosslinked on the analyte sensing constituent according to art acceptedprotocols.

Adhesion Promoting Constituent

The electrochemical sensors of the invention can include one or moreadhesion promoting (AP) constituents (see, e.g. element 414 in FIG. 12).The term “adhesion promoting constituent” is used herein according toart accepted terminology and refers to a constituent that includesmaterials selected for their ability to promote adhesion betweenadjoining constituents in the sensor. Typically, the adhesion promotingconstituent is disposed between the analyte sensing constituent and theanalyte modulating constituent. Typically, the adhesion promotingconstituent is disposed between the optional protein constituent and theanalyte modulating constituent. The adhesion promoter constituent can bemade from any one of a wide variety of materials known in the art tofacilitate the bonding between such constituents and can be applied byany one of a wide variety of methods known in the art. Typically, theadhesion promoter constituent comprises a silane compound such asγ-aminopropyltrimethoxysilane.

Analyte Modulating Constituent

The electrochemical sensors of the invention include an analytemodulating constituent disposed on the sensor (see, e.g. element 412 inFIG. 12). The term “analyte modulating constituent” is used hereinaccording to art accepted terminology and refers to a constituent thattypically forms a membrane on the sensor that operates to modulate thediffusion of one or more analytes, such as glucose, through theconstituent. In certain embodiments of the invention, the analytemodulating constituent is an analyte-limiting membrane which operates toprevent or restrict the diffusion of one or more analytes, such asglucose, through the constituents. In other embodiments of theinvention, the analyte-modulating constituent operates to facilitate thediffusion of one or more analytes, through the constituents. Optionallysuch analyte modulating constituents can be formed to prevent orrestrict the diffusion of one type of molecule through the constituent(e.g. glucose), while at the same time allowing or even facilitating thediffusion of other types of molecules through the constituent (e.g. O₂).

With respect to glucose sensors, in known enzyme electrodes, glucose andoxygen from blood, as well as some interferants, such as ascorbic acidand uric acid, diffuse through a primary membrane of the sensor. As theglucose, oxygen and interferants reach the analyte sensing constituent,an enzyme, such as glucose oxidase, catalyzes the conversion of glucoseto hydrogen peroxide and gluconolactone. The hydrogen peroxide maydiffuse back through the analyte modulating constituent, or it maydiffuse to an electrode where it can be reacted to form oxygen and aproton to produce a current that is proportional to the glucoseconcentration. The analyte modulating sensor membrane assembly servesseveral functions, including selectively allowing the passage of glucosetherethrough (see, e.g. U.S. Patent Application No. 2011-0152654).

Cover Constituent

The electrochemical sensors of the invention include one or more coverconstituents which are typically electrically insulating protectiveconstituents (see, e.g. element 406 in FIG. 12). Typically, such coverconstituents can be in the form of a coating, sheath or tube and aredisposed on at least a portion of the analyte modulating constituent.Acceptable polymer coatings for use as the insulating protective coverconstituent can include, but are not limited to, non-toxic biocompatiblepolymers such as silicone compounds, polyimides, biocompatible soldermasks, epoxy acrylate copolymers, or the like. Further, these coatingscan be photo-imageable to facilitate photolithographic forming ofapertures through to the conductive constituent. A typical coverconstituent comprises spun on silicone. As is known in the art, thisconstituent can be a commercially available RTV (room temperaturevulcanized) silicone composition. A typical chemistry in this context ispolydimethyl siloxane (acetoxy based).

Multilayered Sensor Stacks

An embodiment of the invention having a layered stack of constituents isshown in FIG. 12. FIG. 12 illustrates a cross-section of a typicalsensor embodiment 400 of the present invention that includesconstituents discussed above. This sensor embodiment is formed from aplurality of components that are typically in the form of layers ofvarious conductive and non-conductive constituents disposed on eachother according to art accepted methods and/or the specific methods ofthe invention disclosed herein. The components of the sensor aretypically characterized herein as layers because, for example, it allowsfor a facile characterization of the sensor structure shown in FIG. 12.Artisans will understand however, that in certain embodiments of theinvention, the sensor constituents are combined such that multipleconstituents form one or more heterogeneous layers. In this context,those of skill in the art understand that the ordering of the layeredconstituents can be altered in various embodiments of the invention.

The embodiment shown in FIG. 12 includes a base substrate layer 402 tosupport the sensor 400. The base substrate layer 402 can be made of amaterial such as a metal and/or a ceramic and/or a polymeric substrate,which may be self-supporting or further supported by another material asis known in the art. Embodiments of the invention include a conductivelayer 404 which is disposed on and/or combined with the base substratelayer 402. Typically, the conductive layer 404 comprises one or moreelectrically conductive elements that function as electrodes. Anoperating sensor 400 typically includes a plurality of electrodes suchas a working electrode, a counter electrode and a reference electrode.Other embodiments may also include a plurality of working and/or counterand/or reference electrodes and/or one or more electrodes that performsmultiple functions, for example one that functions as both as areference and a counter electrode.

As discussed in detail below, the base layer 402 and/or conductive layer404 can be generated using many known techniques and materials. Incertain embodiments of the invention, the electrical circuit of thesensor is defined by etching the disposed conductive layer 404 into adesired pattern of conductive paths. A typical electrical circuit forthe sensor 400 comprises two or more adjacent conductive paths withregions at a proximal end to form contact pads and regions at a distalend to form sensor electrodes. An electrically insulating cover layer406 such as a polymer coating can be disposed on portions of the sensor400. Acceptable polymer coatings for use as the insulating protectivecover layer 406 can include, but are not limited to, non-toxicbiocompatible polymers such as silicone compounds, polyimides,biocompatible solder masks, epoxy acrylate copolymers, or the like. Inthe sensors of the present invention, one or more exposed regions orapertures 408 can be made through the cover layer 406 to open theconductive layer 404 to the external environment and to, for example,allow an analyte such as glucose to permeate the layers of the sensorand be sensed by the sensing elements. Apertures 408 can be formed by anumber of techniques, including laser ablation, tape masking, chemicalmilling or etching or photolithographic development or the like. Incertain embodiments of the invention, during manufacture, a secondaryphotoresist can also be applied to the protective layer 406 to definethe regions of the protective layer to be removed to form theaperture(s) 408. The exposed electrodes and/or contact pads can alsoundergo secondary processing (e.g. through the apertures 408), such asadditional plating processing, to prepare the surfaces and/or strengthenthe conductive regions.

In the sensor configuration shown in FIG. 12, an analyte sensing layer410 is disposed on one or more of the exposed electrodes of theconductive layer 404. Typically, the analyte sensing layer 410 is anenzyme layer. Most typically, the analyte sensing layer 410 comprises anenzyme capable of producing and/or utilizing oxygen and/or hydrogenperoxide, for example the enzyme glucose oxidase. Optionally the enzymein the analyte sensing layer is combined with a second carrier proteinsuch as human serum albumin, bovine serum albumin or the like. In anillustrative embodiment, an oxidoreductase enzyme such as glucoseoxidase in the analyte sensing layer 410 reacts with glucose to producehydrogen peroxide, a compound which then modulates a current at anelectrode. As this modulation of current depends on the concentration ofhydrogen peroxide, and the concentration of hydrogen peroxide correlatesto the concentration of glucose, the concentration of glucose can bedetermined by monitoring this modulation in the current. In a specificembodiment of the invention, the hydrogen peroxide is oxidized at aworking electrode which is an anode (also termed herein the anodicworking electrode), with the resulting current being proportional to thehydrogen peroxide concentration. Such modulations in the current causedby changing hydrogen peroxide concentrations can be monitored by any oneof a variety of sensor detector apparatuses such as a universal sensoramperometric biosensor detector or one of the other variety of similardevices known in the art such as glucose monitoring devices produced byMedtronic Diabetes.

In embodiments of the invention, the analyte sensing layer 410 can beapplied over portions of the conductive layer or over the entire regionof the conductive layer. Typically, the analyte sensing layer 410 isdisposed on the working electrode which can be the anode or the cathode.Optionally, the analyte sensing layer 410 is also disposed on a counterand/or reference electrode. Methods for generating a thin analytesensing layer 410 include brushing the layer onto a substrate (e.g. thereactive surface of a platinum black electrode), as well as spin coatingprocesses, dip and dry processes, low shear spraying processes, ink-jetprinting processes, silk screen processes and the like. In certainembodiments of the invention, brushing is used to: (1) allow for aprecise localization of the layer; and (2) push the layer deep into thearchitecture of the reactive surface of an electrode (e.g. platinumblack produced by an electrodeposition process).

Typically, the analyte sensing layer 410 is coated and or disposed nextto one or more additional layers. Optionally, the one or more additionallayers includes a protein layer 416 disposed upon the analyte sensinglayer 410. Typically, the protein layer 416 comprises a protein such ashuman serum albumin, bovine serum albumin or the like. Typically, theprotein layer 416 comprises human serum albumin. In some embodiments ofthe invention, an additional layer includes an analyte modulating layer412 that is disposed above the analyte sensing layer 410 to regulateanalyte contact with the analyte sensing layer 410. For example, theanalyte modulating membrane layer 412 can comprise a glucose limitingmembrane, which regulates the amount of glucose that contacts an enzymesuch as glucose oxidase that is present in the analyte sensing layer.Such glucose limiting membranes can be made from a wide variety ofmaterials known to be suitable for such purposes, e.g., siliconecompounds such as polydimethyl siloxanes, polyurethanes, polyureacellulose acetates, Nafion, polyester sulfonic acids (e.g. Kodak AQ),hydrogels or any other suitable hydrophilic membranes known to thoseskilled in the art.

In certain embodiments of the invention, an adhesion promoter layer 414is disposed between the analyte modulating layer 412 and the analytesensing layer 410 as shown in FIG. 12 in order to facilitate theircontact and/or adhesion. In a specific embodiment of the invention, anadhesion promoter layer 414 is disposed between the analyte modulatinglayer 412 and the protein layer 416 as shown in FIG. 12 in order tofacilitate their contact and/or adhesion. The adhesion promoter layer414 can be made from any one of a wide variety of materials known in theart to facilitate the bonding between such layers. Typically, theadhesion promoter layer 414 comprises a silane compound. In alternativeembodiments, protein or like molecules in the analyte sensing layer 410can be sufficiently crosslinked or otherwise prepared to allow theanalyte modulating membrane layer 412 to be disposed in direct contactwith the analyte sensing layer 410 in the absence of an adhesionpromoter layer 414.

C. Typical System Embodiments of the Invention

A specific illustrative system embodiment consists of a glucose sensorcomprising a folded base architecture as disclosed herein, atransmitter, a recorder and receiver and a glucose meter. In thissystem, radio signals from the transmitter can be sent to the pumpreceiver at regular time periods (e.g. every 5 minutes) to providereal-time sensor glucose (SG) values. Values/graphs can be displayed ona monitor of the pump receiver so that a user can self monitor bloodglucose and deliver insulin using their own insulin pump. Typically, thesensor systems disclosed herein can communicate with a other medicaldevices/systems via a wired or wireless connection. Wirelesscommunication can include for example the reception of emitted radiationsignals as occurs with the transmission of signals via RF telemetry,infrared transmissions, optical transmission, sonic and ultrasonictransmissions and the like. Optionally, the device is an integral partof a medication infusion pump (e.g. an insulin pump). Typically, in suchdevices, the physiological characteristic values includes a plurality ofmeasurements of blood glucose.

FIG. 10 provides a perspective view of one generalized embodiment ofsubcutaneous sensor insertion system that can be adapted for use withthe folded sensor structures disclosed herein and a block diagram of asensor electronics device according to one illustrative embodiment ofthe invention. Additional elements typically used with such sensorsystem embodiments are disclosed for example in U.S. Patent ApplicationNo. 20070163894, the contents of which are incorporated by reference.FIG. 10 provides a perspective view of a telemetered characteristicmonitor system 1, including a subcutaneous sensor set 10 provided forsubcutaneous placement of an active portion of a flexible sensor 12, orthe like, at a selected site in the body of a user. The subcutaneous orpercutaneous portion of the sensor set 10 includes a hollow, slottedinsertion needle 14 having a sharpened tip 44, and a cannula 16. Insidethe cannula 16 is a sensing portion 18 of the sensor 12 to expose one ormore sensor electrodes 20 to the user's bodily fluids through a window22 formed in the cannula 16. The folded base architecture is designed sothat the sensing portion 18 is joined to a connection portion 24 thatterminates in conductive contact pads, or the like, which are alsoexposed through one of the insulative layers. The connection portion 24and the contact pads are generally adapted for a direct wired electricalconnection to a suitable monitor 200 coupled to a display 214 formonitoring a user's condition in response to signals derived from thesensor electrodes 20. The connection portion 24 may be convenientlyconnected electrically to the monitor 200 or a characteristic monitortransmitter 200 by a connector block 28 (or the like) as shown anddescribed in U.S. Pat. No. 5,482,473, entitled FLEX CIRCUIT CONNECTOR,which is incorporated by reference.

As shown in FIG. 10, in accordance with embodiments of the presentinvention, subcutaneous sensor set 10 may be configured or formed towork with either a wired or a wireless characteristic monitor system.The proximal part of the sensor 12 is mounted in a mounting base 30adapted for placement onto the skin of a user. The mounting base 30 canbe a pad having an underside surface coated with a suitable pressuresensitive adhesive layer 32, with a peel-off paper strip 34 normallyprovided to cover and protect the adhesive layer 32, until the sensorset 10 is ready for use. The mounting base 30 includes upper and lowerlayers 36 and 38, with the connection portion 24 of the flexible sensor12 being sandwiched between the layers 36 and 38. The connection portion24 has a forward section joined to the active sensing portion 18 of thesensor 12, which is folded angularly to extend downwardly through a bore40 formed in the lower base layer 38. Optionally, the adhesive layer 32(or another portion of the apparatus in contact with in vivo tissue)includes an anti-inflammatory agent to reduce an inflammatory responseand/or anti-bacterial agent to reduce the chance of infection. Theinsertion needle 14 is adapted for slide-fit reception through a needleport 42 formed in the upper base layer 36 and through the lower bore 40in the lower base layer 38. After insertion, the insertion needle 14 iswithdrawn to leave the cannula 16 with the sensing portion 18 and thesensor electrodes 20 in place at the selected insertion site. In thisembodiment, the telemetered characteristic monitor transmitter 200 iscoupled to a sensor set 10 by a cable 402 through a connector 104 thatis electrically coupled to the connector block 28 of the connectorportion 24 of the sensor set 10.

In the embodiment shown in FIG. 10, the telemetered characteristicmonitor 400 includes a housing 106 that supports a printed circuit board108, batteries 110, antenna 112, and the cable 202 with the connector104. In some embodiments, the housing 106 is formed from an upper case114 and a lower case 116 that are sealed with an ultrasonic weld to forma waterproof (or resistant) seal to permit cleaning by immersion (orswabbing) with water, cleaners, alcohol or the like. In someembodiments, the upper and lower case 114 and 116 are formed from amedical grade plastic. However, in alternative embodiments, the uppercase 114 and lower case 116 may be connected together by other methods,such as snap fits, sealing rings, RTV (silicone sealant) and bondedtogether, or the like, or formed from other materials, such as metal,composites, ceramics, or the like. In other embodiments, the separatecase can be eliminated and the assembly is simply potted in epoxy orother moldable materials that is compatible with the electronics andreasonably moisture resistant. As shown, the lower case 116 may have anunderside surface coated with a suitable pressure sensitive adhesivelayer 118, with a peel-off paper strip 120 normally provided to coverand protect the adhesive layer 118, until the sensor set telemeteredcharacteristic monitor transmitter 200 is ready for use.

In the illustrative embodiment shown in FIG. 10, the subcutaneous sensorset 10 facilitates accurate placement of a flexible thin filmelectrochemical sensor 12 of the type used for monitoring specific bloodparameters representative of a user's condition. The sensor 12 monitorsglucose levels in the body, and may be used in conjunction withautomated or semi-automated medication infusion pumps of the external orimplantable type as described in U.S. Pat. No. 4,562,751; 4,678,408;4,685,903 or 4,573,994, to control delivery of insulin to a diabeticpatient.

In the illustrative embodiment shown in FIG. 10, the sensor electrodes10 may be used in a variety of sensing applications and may beconfigured in a variety of positions on a folded base structure andfurther be formed to include materials that allow a wide variety offunctions. For example, the sensor electrodes 10 may be used inphysiological parameter sensing applications in which some type ofbiomolecule is used as a catalytic agent. For example, the sensorelectrodes 10 may be used in a glucose and oxygen sensor having aglucose oxidase enzyme catalyzing a reaction with the sensor electrodes20. The sensor electrodes 10, along with a biomolecule or some othercatalytic agent, may be placed in a human body in a vascular ornon-vascular environment. For example, the sensor electrodes 20 andbiomolecule may be placed in a vein and be subjected to a blood stream,or may be placed in a subcutaneous or peritoneal region of the humanbody.

In the embodiment of the invention shown in FIG. 10, the monitor ofsensor signals 200 may also be referred to as a sensor electronicsdevice 200. The monitor 200 may include a power source, a sensorinterface, processing electronics (i.e. a processor), and dataformatting electronics. The monitor 200 may be coupled to the sensor set10 by a cable 402 through a connector that is electrically coupled tothe connector block 28 of the connection portion 24. In an alternativeembodiment, the cable may be omitted. In this embodiment of theinvention, the monitor 200 may include an appropriate connector fordirect connection to the connection portion 104 of the sensor set 10.The sensor set 10 may be modified to have the connector portion 104positioned at a different location, e.g., on top of the sensor set tofacilitate placement of the monitor 200 over the sensor set.

As noted above, embodiments of the sensor elements and sensors can beoperatively coupled to a variety of other system elements typically usedwith analyte sensors (e.g. structural elements such as piercing members,insertion sets and the like as well as electronic components such asprocessors, monitors, medication infusion pumps and the like), forexample to adapt them for use in various contexts (e.g. implantationwithin a mammal). One embodiment of the invention includes a method ofmonitoring a physiological characteristic of a user using an embodimentof the invention that includes an input element capable of receiving asignal from a sensor that is based on a sensed physiologicalcharacteristic value of the user, and a processor for analyzing thereceived signal. In typical embodiments of the invention, the processordetermines a dynamic behavior of the physiological characteristic valueand provides an observable indicator based upon the dynamic behavior ofthe physiological characteristic value so determined. In someembodiments, the physiological characteristic value is a measure of theconcentration of blood glucose in the user. In other embodiments, theprocess of analyzing the received signal and determining a dynamicbehavior includes repeatedly measuring the physiological characteristicvalue to obtain a series of physiological characteristic values in orderto, for example, incorporate comparative redundancies into a sensorapparatus in a manner designed to provide confirmatory information onsensor function, analyte concentration measurements, the presence ofinterferences and the like.

FIG. 11 shows a schematic of a potentiostat that may be used to measurecurrent in embodiments of the present invention. As shown in FIG. 11, apotentiostat 300 may include an op amp 310 that is connected in anelectrical circuit so as to have two inputs: Vset and Vmeasured. Asshown, Vmeasured is the measured value of the voltage between areference electrode and a working electrode. Vset, on the other hand, isthe optimally desired voltage across the working and referenceelectrodes. The current between the counter and reference electrode ismeasured, creating a current measurement (Isig) that is output from thepotentiostat.

Embodiments of the invention include devices which process display datafrom measurements of a sensed physiological characteristic (e.g. bloodglucose concentrations) in a manner and format tailored to allow a userof the device to easily monitor and, if necessary, modulate thephysiological status of that characteristic (e.g. modulation of bloodglucose concentrations via insulin administration). An illustrativeembodiment of the invention is a device comprising a sensor inputcapable of receiving a signal from a sensor, the signal being based on asensed physiological characteristic value of a user; a memory forstoring a plurality of measurements of the sensed physiologicalcharacteristic value of the user from the received signal from thesensor; and a display for presenting a text and/or graphicalrepresentation of the plurality of measurements of the sensedphysiological characteristic value (e.g. text, a line graph or the like,a bar graph or the like, a grid pattern or the like or a combinationthereof). Typically, the graphical representation displays real timemeasurements of the sensed physiological characteristic value. Suchdevices can be used in a variety of contexts, for example in combinationwith other medical apparatuses. In some embodiments of the invention,the device is used in combination with at least one other medical device(e.g. a glucose sensor).

An illustrative system embodiment consists of a glucose sensor, atransmitter and pump receiver and a glucose meter. In this system, radiosignals from the transmitter can be sent to the pump receiver every 5minutes to provide real-time sensor glucose (SG) values. Values/graphsare displayed on a monitor of the pump receiver so that a user can selfmonitor blood glucose and deliver insulin using their own insulin pump.Typically, an embodiment of device disclosed herein communicates with asecond medical device via a wired or wireless connection. Wirelesscommunication can include for example the reception of emitted radiationsignals as occurs with the transmission of signals via RF telemetry,infrared transmissions, optical transmission, sonic and ultrasonictransmissions and the like. Optionally, the device is an integral partof a medication infusion pump (e.g. an insulin pump). Typically, in suchdevices, the physiological characteristic values include a plurality ofmeasurements of blood glucose.

While the analyte sensor and sensor systems disclosed herein aretypically designed to be implantable within the body of a mammal, theinventions disclosed herein are not limited to any particularenvironment and can instead be used in a wide variety of contexts, forexample for the analysis of most in vivo and in vitro liquid samplesincluding biological fluids such as interstitial fluids, whole-blood,lymph, plasma, serum, saliva, urine, stool, perspiration, mucus, tears,cerebrospinal fluid, nasal secretion, cervical or vaginal secretion,semen, pleural fluid, amniotic fluid, peritoneal fluid, middle earfluid, joint fluid, gastric aspirate or the like. In addition, solid ordesiccated samples may be dissolved in an appropriate solvent to providea liquid mixture suitable for analysis.

It is to be understood that this invention is not limited to theparticular embodiments described, as such may, of course, vary. It isalso to be understood that the terminology used herein is for thepurpose of describing particular embodiments only, and is not intendedto be limiting, since the scope of the present invention will be limitedonly by the appended claims. In the description of the preferredembodiment, reference is made to the accompanying drawings which form apart hereof, and in which is shown by way of illustration a specificembodiment in which the invention may be practiced. It is to beunderstood that other embodiments may be utilized and structural changesmay be made without departing from the scope of the present invention.

The descriptions and specific examples, while indicating someembodiments of the present invention are given by way of illustrationand not limitation. Many changes and modifications within the scope ofthe present invention may be made without departing from the spiritthereof, and the invention includes all such modifications.

The invention claimed is:
 1. A method of sensing an analyte within amammal comprising: (a) implanting an analyte sensor apparatus within themammal, the analyte sensor apparatus comprising: a base substratecomprising a planar sheet of a flexible material adapted to transitionfrom a first configuration to a second configuration when the basesubstrate is folded to form a fixed bend; a working electrode, a counterelectrode and a reference electrode disposed upon a first surface of thebase substrate; a plurality of contact pads disposed upon the firstsurface of the base substrate; a plurality of electrical conduitsdisposed upon the first surface of the base substrate, wherein theplurality of electrical conduits are adapted to transmit electricalsignals between the working electrode, the counter electrode or thereference electrode and the plurality of contact pads separated by thefixed bend; an analyte sensing layer comprising glucose oxidase disposedover the working electrode, wherein the analyte sensing layer detectablyalters the electrical current at the working electrode in the presenceof an analyte; and an analyte modulating layer disposed over the analytesensing layer, wherein the analyte modulating layer modulates thediffusion of analyte therethrough; wherein: the base substrate comprisesthe fixed bend so as to form a configuration characterized in that: atleast one working electrode, counter electrode or reference electrode isdisposed on a first side of the fixed bend; and at least one workingelectrode, counter electrode or reference electrode is disposed on asecond side of the fixed bend; (b) sensing an alteration in current atthe working electrode in the presence of the analyte; and (c)correlating the alteration in current with the presence of the analyte,so that the analyte is sensed.
 2. The method of claim 1, wherein thefixed bend configures the base substrate in an orientation such that atleast one electrode on the first side of the fixed bend and at least oneelectrode on the second side of the fixed bend face opposite directions.3. The method of claim 1, wherein the base substrate comprises at leastone of: a demarcation, a perforation, or a kiss cut disposed in an areaat which the base substrate is folded.
 4. The method of claim 1,wherein: the analyte sensor apparatus does not comprise a housing thatsurrounds the analyte sensor apparatus; or the base substrate does notcomprise an electrical via.
 5. The method of claim 2, wherein theanalyte sensor apparatus further comprises: a processor; acomputer-readable program code having instructions, which when executedcause the processor to: assess electrochemical signal data obtained fromthe working electrode; and compute analyte concentrations based upon theelectrochemical signal data obtained from the working electrode.
 6. Themethod of claim 1, wherein the analyte is glucose.
 7. The method ofclaim 1, wherein the analyte sensor apparatus is implanted within theinterstitial space of a diabetic.
 8. A method of sensing an analytewithin a diabetic patient comprising: (a) implanting an analyte sensorapparatus within an interstitial space of the diabetic patient, theanalyte sensor apparatus comprising: a base substrate comprising aplanar sheet of a flexible material adapted to transition from a firstconfiguration to a second configuration when the base substrate isfolded to form a fixed bend; a working electrode, a counter electrodeand a reference electrode disposed upon a first surface of the basesubstrate; a plurality of contact pads disposed upon the first surfaceof the base substrate; a plurality of electrical conduits disposed uponthe first surface of the base substrate, wherein the plurality ofelectrical conduits are adapted to transmit electrical signals betweenthe working electrode, the counter electrode or the reference electrodeand the plurality of contact pads separated by the fixed bend; ananalyte sensing layer comprising glucose oxidase disposed over theworking electrode, wherein the analyte sensing layer detectably altersthe electrical current at the working electrode in the presence of ananalyte; and an analyte modulating layer disposed over the analytesensing layer, wherein the analyte modulating layer modulates thediffusion of analyte therethrough; wherein: the base substrate comprisesthe fixed bend so as to form a configuration characterized in that: atleast one working electrode, counter electrode or reference electrode isdisposed on a first side of the fixed bend; and at least one workingelectrode, counter electrode or reference electrode is disposed on asecond side of the fixed bend; (b) sensing an alteration in current atthe working electrode in the presence of the analyte; and (c)correlating the alteration in current with the presence of the analyte,so that the analyte is sensed.
 9. The method of claim 8, wherein thefixed bend configures the base substrate in an orientation such that theworking electrode on the first side of the fixed bend and the counterelectrode on the second side of the fixed bend face opposite directions.10. The method of claim 8, wherein the base substrate comprises at leastone of: a demarcation, a perforation, or a kiss cut disposed in an areaat which the base substrate is folded.
 11. The method of claim 8,wherein the base substrate comprises: a rectangular body; a firstlongitudinal arm extending outward from the rectangular body; and asecond longitudinal arm extending outward from the rectangular body;wherein the first longitudinal arm and the second longitudinal arm areparallel to each other.
 12. The method of claim 11, further comprising alocking member disposed on the base substrate and adapted to inhibitmovement of the first longitudinal arm or the second longitudinal arm.13. The method of claim 8, wherein: the analyte sensor apparatus doesnot comprise a housing that surrounds the analyte sensor apparatus; orthe base substrate does not comprise an electrical via.
 14. The methodof claim 8, wherein the analyte sensor apparatus further comprises: aprocessor; a computer-readable program code having instructions, whichwhen executed cause the processor to: assess electrochemical signal dataobtained from the working electrode; and compute analyte concentrationsbased upon the electrochemical signal data obtained from the workingelectrode.
 15. The method of claim 8, wherein the analyte is glucose.16. The method of claim 8, wherein the analyte sensing layer comprisesglucose oxidase.